We have shown that transcriptional noise is
well predicted by molecularly detailed models
for the two most common promoter architectures in E. coli as the various genetic knobs are
tuned. This agreement is not the result of fitting
theory curves to data, because the predicted curves
are generated using physical parameter values
reported elsewhere in the literature and in that
sense are zero-parameter predictions. Earlier reports of “bursty” transcription (5, 21) are based
on the observation that the Fano factor is greater
than 1 for constitutive mRNA production (as well
as direct kinetic measurements). Various explanatory hypotheses have been proposed, including
transcriptional silencing via DNA condensation by nucleoid proteins (22), negative supercoil-ing induced by transcription, or the formation
of long-lived “dead-end” initiation complexes
(23). Although our data do not rule out these
hypotheses, we find that extrinsic noise is sufficient to explain the deviation from Fano = 1 in
our constitutive expression data (Fig. 2B). Thus,
we find no need to invoke alternative hypotheses to explain the observed “burstiness” of constitutive transcription.
Many interesting earlier experiments make
it difficult to interpret differences between promoters and induction conditions in terms of
distinct physical parameters because of the wide
variety of promoter architectures in play as well
as the diverse mechanisms of induction. We have
instead taken a “synthetic biology” approach of
building promoters from the ground up. By directly controlling aspects of the promoter architecture, our goal has been to directly relate changes
in promoter architecture to changes in observed
gene expression variability. We believe that this
work has demonstrated that mutations in regulatory DNA can alter gene expression noise. This
suggests that gene expression noise may be a
tunable property subject to evolutionary selection pressure, as mutations in regulatory DNA
could provide greater fitness by increasing (or
decreasing) variability. Demonstrating the relevance of this hypothesis in natural environments
remains an ongoing challenge.
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We thank H. J. Lee, C. Wiggins, Y. Lin, X. Zhu, F. Weinert,
M. Rydenfelt, R. Milo, H. Garcia, N. Belliveau, and J. Sheung for
useful discussions. Supported by NIH grants DP1 OD000217
(Directors Pioneer Award), R01 GM085286, and 1 U54 CA143869
(Northwestern PSOC Center); La Fondation Pierre Gilles de
Gennes (R.P.); and the Donna and Benjamin M. Rosen Center
for Bioengineering at Caltech (D.L.J.). Raw microscopy image
data are archived in the Phillips laboratory at Caltech and
are available upon request.
Materials and Methods
Figs. S1 to S11
Tables S1 to S3
28 April 2014; accepted 4 November 2014
Detection of self-reactive CD8+
T cells with an anergic phenotype
in healthy individuals
Yuka Maeda,1 Hiroyoshi Nishikawa,1 Daisuke Sugiyama,1 Danbee Ha,1
Masahide Hamaguchi,1 Takuro Saito,1 Megumi Nishioka,1,2 James B. Wing,1
Dennis Adeegbe,1 Ichiro Katayama,2 Shimon Sakaguchi1*
Immunological tolerance to self requires naturally occurring regulatory T (Treg) cells. Yet
how they stably control autoimmune T cells remains obscure. Here, we show that Treg cells
can render self-reactive human CD8+ T cells anergic (i.e., hypoproliferative and cytokine
hypoproducing upon antigen restimulation) in vitro, likely by controlling the costimulatory
function of antigen-presenting cells. Anergic T cells were naïve in phenotype, lower than
activated T cells in T cell receptor affinity for cognate antigen, and expressed several
coinhibitory molecules, including cytotoxic T lymphocyte–associated antigen-4 (CTLA-4).
Using these criteria, we detected in healthy individuals anergic T cells reactive with a skin
antigen targeted in the autoimmune disease vitiligo. Collectively, our results suggest that
Treg cell–mediated induction of anergy in autoimmune T cells is important for maintaining
Naturally occurring CD25+CD4+ regulatory T (Treg) cells, which specifically express the transcription factor FoxP3, actively main- tain immunological self-tolerance and ho- meostasis (1). Developmental or functional
anomalies of natural Treg cells can cause autoimmune diseases (such as type I diabetes), allergy, and immunopathological diseases (such as
inflammatory bowel disease) (1). How Treg cells
effectively control potentially hazardous self-reactive T cells in humans remains an open question. In particular, it is unknown whether Treg
cell–mediated suppression for a limited period
has a critical long-lasting effect on cell fate and antigen reactivity of autoimmune T cells.
To address this issue, we examined proliferation,
cytokine production, and cell fate of antigen-
specific CD8+ T cells in peripheral blood mono-
nuclear cells (PBMCs) from healthy individuals
stimulated in vitro with self-antigen peptide in the
presence or absence of natural FoxP3+CD25+CD4+
Treg cells. Melan-A (also known as MART-1) used
in the experiments is a self-antigen expressed by
normal melanocytes and some melanoma cells
and targeted in vitiligo vulgaris, an autoimmune
disease of the skin (2–5). In the absence of Treg
cells, Melan-A–specific CD8+ T cells [detectable
by major histocompatibility complex (MHC) tetra-
mers and peptide tetramers] expanded over 10
days from very few cells to a sizable fraction
when cultured with peptide-pulsed autologous
antigen-presenting cells (APCs) (Fig. 1A) (6). Nat-
ural Treg cells, which appeared to be activated by
endogenous self-peptides and class II MHC on
autologous APCs (7–9), suppressed the expansion
of Melan-A tetramer–positive (Tet+) CD8+ T cells
in a dose-dependent manner. Similar stimulation
with irrelevant peptide NY-ESO-1, another self-
and tumor antigen, failed to induce Melan-
ATet+CD8+ T cells. In cultures containing Treg cells,
1Experimental Immunology, Immunology Frontier Research
Center (IFReC-WPI), Osaka University, Osaka 565-0871,
Japan. 2Department of Dermatology, Graduate School of
Medicine, Osaka University, Osaka 565-0871, Japan.
*Corresponding author. E-mail: email@example.com
(S.S.); firstname.lastname@example.org (H.N.)